The present invention relates to the protein purification arts and more specifically to methods for producing highly purified follicle stimulating hormone (FSH) to high purity FSH compositions.
Follicle stimulating hormone (FSH, or follitropin) is a pituitary heterodimeric glycoprotein hormone synthesized and released from gonadotrope cells of the anterior pituitary gland. As a circulating hormone, FSH interacts with high affinity with receptor molecules on the surface of granulosa cells in the ovary. This interaction evokes a series of intracellular events, including increasing intracellular levels of the second messenger, cyclic AMP, and elicitation of a steroidogenic response by the granulosa cells resulting in estrogen production. Local estrogen and FSH stimulation promote the growth and maturation of ovarian follicles.
The amount of circulating FSH is dependent upon several other endocrine and neural factors. Gonadotropin releasing hormone (GnRH) is a peptide elaborated by hypothalamic neurons. Released GnRH interacts with receptors on pituitary gonadotrope cells to control the synthesis and release of FSH and leutenizing hormone (LH) from the anterior pituitary gland. FSH secretion also is affected by circulating levels of steroid hormones. The steroidogenic response of granulosa cells to FSH results in gradually increasing estradiol levels. When serum estradiol reaches a critical level, it triggers a large increase in the rate of LH and FSH release from the anterior pituitary. The resultant LH surge induces ovulation and luteinization of granulosa cells. Progesterone is released from the corpus luteum following ovulation and this steroid prepares the uterus for implantation of the fertilized ovum. Elevated levels of estrogens and progesterone exert a negative feedback inhibition at hypothalamic sites to lower FSH and LH synthesis and release. Hence, the effects of steroids on gonadotropin release depend on the circulating levels; at low levels of estrogen, FSH and LH are positively regulated while higher levels result in negative feedback inhibition.
In males, FSH induces spermatogenesis through a proliferative effect on spermatocytes. Sperm production also requires testosterone, which is under positive regulatory control by LH.
An apparent paradox of the above-described hormonal control process is the production of both LH and FSH by the gonadotrope cell while GnRH serves as a positive regulatory agent for both hormones. Work within the last 10 years suggests that the peptides activin, follistatin, art and inhibin selectively regulate FSH secretion from the anterior pituitary gland. FSH synthesis and release are activated by activin, while inhibin and follistatin have negative feedback effects. The inhibitory effect of follistatin is thought to be mediated by its high-affinity binding to activin and blockage of its biological activity. There is evidence for both autocrine and paracrine local regulatory effects of these peptides and for feedback effects of inhibin released from gonadal tissue. Both inhibin and activin are structurally related and are members of the diverse transforming growth factor beta family of peptides. Study of the physiological roles of activin, follistatin, and inhibin is a current area of active research (reviewed by Knight, 1996).
FSH has been used extensively as a drug to treat human infertility by induction of follicular development in females. Earlier products were crude preparations of LH and FSH, i.e., Pergonal(copyright). More recently developed products contain purer FSH preparations. Metrodin(copyright) has low levels of LH and the FSH specific activity is about 100 IU of FSH per mg total protein. This drug requires intramuscular injections every day for 5 to 7 days, followed by a single injection of human chorionic gonadotropin (hCG) to induce ovulation. A recent advance is Fertinex(copyright) which is affinity-purified FSH from human menopausal gonadotrophin (hMG). This product exhibits a FSH potency of 8500 to 13,500 IU of FSH per mg total protein at 95% purity as reported by the manufacturer. The high purity of Fertinex(copyright) allows delivery by subcutaneous injection, which can be administered at home. Following administration of Fertinex(copyright), hCG is used for induction of ovulation. Depending on the dosage of FSH administered, it may be used to promote in vivo fertility or, at higher dosages, it may be used to induce multiple oocyte formation for in vitro fertilization procedures. Recombinant forms of FSH (Puregon(copyright) and Gonal(copyright)) also are used as fertility drugs; these versions of FSH have potencies and-purities similar to that of Fertinex(copyright).
FSH has been purified from pituitary glands, human postmenopausal urine, and from culture media collected from genetically engineered cells. FSH purification has been an active area of research over the past 30 years. Older methods rely on procedures such as ion exchange chromatography, size exclusion chromatography, polyacrylamide gel electrophoresis, and chromatography on hydroxylapatite. In one method (Roos, et al., 1968) a FSH preparation of 14,000 IU of FSH per mg total protein was obtained from fresh frozen human pituitary glands with an overall recovery of activity of 5.0%. A similar procedure applied to urinary FSH resulted in a preparation of 780 IU of FSH per mg total protein at a 7.7% overall yield.
Because of the similar physicochemical properties of FSH and LH, i.e., similar molecular weight and overlapping isoelectric profiles, affinity chromatography methods have been employed to improve the separation of LH from FSH. Affinity methods also afford the possibility of high purification in a single step (up to 100-fold) thereby reducing the number of steps in a purification method and improving overall yield. The latter is a critical factor in the commercial production of FSH as overall yield is a major determinant of cost. Group-specific affinity adsorbents such as the lectin Concanavalin A or chitosan (Japanese patent number 8,027,181) bind glycoproteins via specific carbohydrate groups. Concanavalin A has been used to characterize microheterogeneity of purified FSH preparations (Chapped, et al., 1983). However, these ligands are ineffective in the separation of two glycoproteins such as FSH and LH.
Immunoaffinity chromatography (IAC) relies upon the specificity of mono- or polyclonal antibodies for capture of specific protein antigens from crude mixtures. Antibodies may first be screened for use in IAC (Bonde, et al., 1991). The selected antibodies are coupled to a chromatographic solid phase, e.g., cross-linked agarose, through covalent bonds, e.g., cyanogen bromide (CNBr) or other coupling chemistries targeting surface amino, hydroxyl, carboxyl or sulfhydryl groups of immunoglobulins to form a solid matrix. Recent coupling methods attempt site-directed immobilization of antibodies in an effort to optimize antigen-binding efficiencies, which are typically low, using classical coupling chemistries. One method of site-directed coupling is through carbohydrate groups of the Fc immunoglobulin region to hydrazide-activated solid supports (Hoffman and O""Shannessy, 1988). The solid phase coupled to antibody then is packed in a chromatography column and equilibrated with buffer for binding to antigen. Mixtures of target protein and contaminants are equilibrated with binding buffer and then applied to the column. Non-adsorbed contaminants are removed by washout with various buffers. Elution occurs by use of chaotropic agents, extremes of pH, changes in ionic strength, etc.. Elution is a critical aspect of IAC since the elution conditions may alter the biological activity of the immobilized antibody or the eluted antigen or both (reviewed by Jack, 1994).
IAC may be used to remove specific contaminants from a crude mixture. This mode first was applied to FSH purification using antibodies to hCG, which through cross reactivity to LH, effectively reduced LH contamination levels (Donini, et al., 1966). Other methods (Jack, et al., 1987 and Great Britain patent number 8,510,177) utilized monoclonal antibody specific to FSH for IAC. The antibody was coupled to CNBr-activated Sepharose 4B. Samples were applied in a buffer of 0.05 M borate, 0.5 M NaCl at pH 8.5 and non-adsorbed contaminants were eluted from the column with 0.05 M borate at pH 8.5. The bound FSH was eluted using 0.1 M glycine, 0.5 M NaCl at pH 3.5. When using a sample containing the glycoprotein-enriched fraction from a side-fraction of human growth hormone obtained from frozen pituitaries, 47% of the applied FSH was recovered from the IAC procedure. The FSH was recovered at a specific activity of 10,000 IU of FSH per mg total protein. It contained 0.0014 IU of LH per IU of FSH and thyroid stimulating hormones (TSH) at 0.93 xcexcIU of TSH per IU of FSH (Jack, et al., 1987). Another IAC method for FSH relies on a monoclonal antibody to FSH that is coupled to Sepharose 4B by divinylsulphone (U.S. Pat. No. 5,128,453). The column and sample were equilibrated with 0.1 M Tris, 0.3 M NaCl at pH 7.5 and the IAC procedure was performed at 4xc2x0 C. In this case, partially purified urinary FSH (hMG) was used as a sample. The sample was applied in the equilibration buffer and nonadsorbed materials were removed by continued washing with this buffer. FSH elution was accomplished by use of high ionic strength alkaline buffers, e.g., 1 M ammonia or other eluents of pH greater than 11 and of ionic strength greater than 0.8 M. The product of IAC was then subject to reverse phase HPLC on a C18 column to generate the final product. While the yield of FSH activity from the IAC step was not given, the final product had a specific biological activity of 6200 IU of FSH per mg total protein (specific biological activity=1.2xc3x97specific immunological activity for this purified material) and had undetectable levels of LH contamination by radio immunoassay (RIA) measurement. No other protein contaminants were detected by SDS-PAGE analysis.
Other researchers have recently reported the use of dye affinity chromatography (DAC) in the purification of FSH from the brushtail possum. Their purification involved several steps including the use of Green A Matrix gel and Red A Matrix gel (Amicon, Inc., Beverly, Mass.) for two sequential DAC procedures. The overall yield of their method was 12% (Moore, et al., 1997).
The present invention provides a process for the purification of FSH by use of DAC. The method utilizes a dye ligand, for example an orange dye ligand, preferably Orange 1 (Prometic Biosciences, Inc., Burtonsville, Md.), which is coupled to cross-linked agarose via triazine coupling chemistry to form a solid matrix. Other dye ligands also may be used, for example, Orange 2, Yellow 2 or Green 1 (Prometic Biosciences, Inc., Burtonsville, Md.). Orange 1 shows strong selectivity to FSH when the binding occurs at low ionic strength and acidic pH, for example, about pH 4.0. Samples containing FSH and excess amounts of LH fail to exhibit significant LH binding under these conditions and LH contamination is conveniently removed by elution with an appropriate washout buffer. FSH then may be eluted from the dye by increasing eluent ionic strength, for example, by use of a linear salt gradient, preferably 0 to 0.6 M NaCl. Alternatively, eluent ionic strength can be increased step-wise to elute the FSH. Other means of eluting or releasing the bound FSH may be used such as, for example, increasing pH or using agents which compete for FSH binding to the dye ligand. The result is FSH product containing only minimal contamination by LH and other unwanted proteins. Residual contamination of FSH by LH and other unwanted proteins may then be removed by, for example, hydrophobic interaction chromatography (HIC) or ion exchange chromatography. The FSH purification methods of the present invention have been used to purify, for example, human pituitary FSH, human urinary FSH, human recombinant FSH, human FSH secreted from gonadotropes, and bovine FSH. The present method may also be used to purify FSH from other species, particularly mammalian, including, for example, equine, porcine, ovine, canine, feline, rat, mouse and monkey.
The present invention operates at high yields ( greater than 95% recovery of FSH activity) and with high purification factors (up to about 50-fold) depending upon the sample used. The method is non-denaturing to FSH and this allows for high overall recoveries in multi-step purification procedures as needed, for example, when FSH is purified from human pituitary glands. Also, the ligand shows minimal ligand leakage and can be regenerated with substantially complete restoration of FSH binding properties. Therefore, the dye may be used for at least 25 cycles prior to loss of effective binding and release of FSH.
The advances of this invention over the prior art include the advantages of affinity chromatography by DAC as compared to IAC. The primary advance is in the ease of elution of bound FSH from the immobilized dye ligand as compared to an immobilized antibody. While very gentle elution conditions are used in the present invention, e.g., sodium chloride gradient at pH 6.0, elution from an IAC column usually involves relatively harsh conditions. One IAC method involves use of an elution buffer at pH 3.5 to elute bound FSH (Jack et al., 1987). This pH has deleterious effects on the immunological activity of human FSH. While use of alkaline pH (U.S. Pat. No. 5,128,453) eliminates the harmful effects of low pH, the effects of alkaline pH on the immobilized antibody are unknown. Even minor effects on the immobilized antibody could tend to decrease the effectiveness of the immunoadsorbant with continued use cycles. In addition, IAC requires highly consistent batches of monoclonal antibody and coupling procedures, which may result in low antigen binding efficiencies. Lower binding capacity would require larger amounts of antibody to bind a given amount of antigen. DAC relies on an inert ligand, which can be manufactured and coupled in a highly reproducible manner.
Another advantage of DAC resins is in sanitization. DAC resins may by sanitized and depyrogenated by treatment with 1.0 N NaOH without effect on chromatographic performance. Such treatment of IAC resins usually results in inactivation of the antibody. Therefore, sanitation of IAC resins is more difficult to achieve. Minimal ligand leakage from DAC resins also results in lower product contamination, while antibody leakage from IAC resins can contaminate the product. For reviews of DAC see: Lowe, et al., (1992) and Garg, et al., (1996).
This invention is intended for use with biological materials, particularly relatively crude mixtures of FSH, LH and other contaminating proteins referred to herein as starting material sample(s) or starting material(s) or sample(s). The examples described in detail below use starting material samples obtained during pituitary hormone purification from human or bovine pituitary glands; hMG prepared from human menopausal urine; FSH derived from primary cultured human gonadotrope cells; and crude preparations of recombinantly produced human FSH (recombinant human FSH). Alternative sources of starting material might include: 1) FSH extracted from the pituitary glands obtained from other species, especially mammalian, for example, equine, porcine, ovine, canine, feline, rat, mouse, and monkey; 2) recombinant FSH or FSH derived from the gonadotropes or gonadotrope cell lines obtained from other species, especially mammalian, for example, bovine, equine, porcine, ovine, canine, feline, rat, mouse, and monkey; 3) genetically or otherwise altered forms of FSH (Szkudlinski, et al., 1996; U.S. Pat. No. 5,338,835) obtained from various species, especially mammalian, for example, human, bovine, equine, porcine, ovine, canine, feline, rat, mouse, and monkey. In any case, the sample is substantially free of TSH, prolactin, and growth hormone and the FSH comprises about 2-5% of the sample. The LH contamination varies according to the sample but may be as high as 200%.
The sample can be prepared for DAC by standard methods of sample preparation including concentration-diafiltration or concentration and desalting using size exclusion chromatography. The sample preferably is equilibrated with sodium acetate with or without leupeptin as a protease inhibitor. More preferably, the sample is equilibrated with about 1 mM to about 5 mM sodium acetate containing about 1 xcexcM to about 2 xcexcM leupeptin at pH 4.0 to 5.5. Even more preferably, the sample is equilibrated with 5 mM sodium acetate at pH 4.0. For longer term storage of these samples, it is preferable to use 1 mM to 5 mM Tris-acetate, more preferably 1 mM, as the equilibration buffer and maintain the sample pH at 7 to 9.5, more preferably pH 9.5. The sample may be stored liquid or frozen in this buffer without appreciable loss of FSH activity. The sample should only be exposed to low pH (e.g., about 4) for relatively short time periods just prior to its application to the DAC column. Longer-term exposure to 5 mM sodium acetate at pH 4.0 can lead to FSH inactivation.
The DAC column is prepared in, preferably, a glass chromatography column of appropriate dimensions for the sample to be used and the target loading volume of the DAC media. In general one of skill in the art will readily determine appropriate volumes of DAC media to use, based on the capacity of the medium to bind FSH. The capacity can be determined by, e.g., incubating aliquots of medium with different volumes of sample, and determining the amount of FSH remaining in the sample. The column is packed with the solid matrix comprising an appropriate dye coupled to the solid phase, preferably Mimetic Orange 1 (Prometic Biosciences, Inc., Burtonsville, Md.; Cat. No. A6XL 0030). The column then is equilibrated with several column volumes, preferably at least 8, of sodium acetate at acidic pH at a linear flow rate of 60-120 cm/hr. Linear flow rate for a cylindrical column is defined as flow rate, expressed as mL/hr, divided by xcfx80r2 where r equals the column radius in cm. The conductivity of the sample is desirably less than 1 mS prior to binding. The sample is at acidic pH, preferably about 4.0, in either sodium acetate (preferably about 5 mM) or Tris-acetate (preferably about 1 mM). An aliquot of the sample is taken for later assay. The sample is applied to the column, preferably at 30 cm/hr or less. Sample application is followed by application of a sodium acetate buffer, at pH 4.0, for several column volumes at about 60 cm/hr. This is followed by washout of non-adsorbed LH and other proteins using a NaH2PO4 buffer at pH about 6.0. Washout with this buffer continues for a total of several column volumes at about 60 cm/hr. FSH is then eluted or released by use of a salt buffer which also contains NaH2PO4 at pH about 6.0. Elution of FSH by use of a salt buffer is done using a linear salt gradient, or alternatively, performed with a step-wise increase in buffer salt concentration. As an alternative to releasing FSH with increasing ionic strength, FSH also can be eluted using increasing pH or agents that compete with FSH binding to the dye ligand.